Rabies vaccine and process for preparation thereof



United States Patent Ofifice 3,423,595 Patented Jan. 21, 1969 915,362US. Cl. 424-89 rm. Cl. A61k 23/00,- C12k 5/00 12 Claims ABSTRACT OF THEDISCLOSURE A process for producing rabies vaccine for immunizing animalsand the rabies vaccine per se in which the ERA strain of rabies virusATCC VR 332 is propagated in live kidney cells obtained from pigs ordogs by tissue cell techniques with a resulting rabies viruscontainingsolution (vaccine) substantially free from contaminants (either viral orbacterial).

Background of invention In the treatment of man, the rabies vaccine nowfrequently used is described as modified Semple vaccine. This vaccineconsists of a suspension of fixed rabies virus prepared from the brainsof animals and inactivated with phenol. In the immunization of animalssuch as cattle, dogs, cats, etc., the rabies vaccine termed Fluryvaccine is used. This vaccine is a live attenuated preparation and isproduced by grOWing a suitable strain of the virus in embryonic chicks.Thus, in either instance, the preparation which is injected consists ofthe virus and of a relatively large amount of the tissue in which thevirus was grown; that is, brain suspension in the case of the Semplevaccine or embryonic chicks in the case of the Flury vaccine. It hasbeen observed that in the case of the Semple vaccine, the daily dose ofrabid brain tissue administered to a human subject may be as much as 80milligrams. The amount of virus protein which is administered is verysmall in comparison. An objection to such vaccines is that a relativelylarge amount of undesirable protein to which the person or animal beingtreated or immunized may be sensitive, is administered with therelatively small amount of active virus. It is well known that anyreaction to these proteins is very painful and may be harmful.

Description of the invention An object of this invention is to provide anew strain of rabies virus which is immunogenic and not pathogenic tothe subject to be immunized against rabies.

Another object of this invention is to provide an immunogenic strain ofrabies which is adapted to grow on tissue cultures and furthermore,which is particularly effective in immunization.

A further object of this invention is to provide a rabies vaccine whichis substantially free of undesirable protein.

An even further object of this invention is to provide a rabies vaccinein which the chance occurrence of a contaminant (either viral orbacterial) has been considerably reduced from that in vaccines producedby other methods.

The vaccine of this invention has been tested in several species ofanimals and shown to be effective to immunize these animals againstrabies. From the results of these tests. it is contemplated that thevaccine may be used to treat man without harmful effect.

Our colleague, Dr. Paul Fenje has adapted a strain of rabies fixed virusdesignated SAD to grow in hamster kidney tissue (Canadian Journal ofMicrobiology 6, 606,

l960). We obtained a sample of virus from Dr. Fenjc and adapted it forgrowth in cells such as dog or pig kidney cells as hereinafterdescribed.

We have successfully grown rabies virus in tissue cul ture technique ingood yields and on a large scale which fulfills the objects of thisinvention. Among the cells which we have used, and which are usuallyavailable at any time of the year, are dog kidney cells and pig kidneycells. Pig kidney cells are preferred partly because pig kidney cellsare available all times of the year and in large quantities. Moreover,in practicing the invention. it is desir able to use cells from aspecies unlikely to be infected with a disease which may be found in thespecies to be vaccinated. it is preferable to use cells from minimaldisease herds.

One method of adapting this strain for growth in kidney cells. forexample, in pig kidney cells. is first to grow it in fertile eggs,preferably for several. such as ten, passages; then, after transferthrough pig kidney cells at least once, we call this virus strain theERA strain of rabies virus. A sample of the ERA strain of virus wasdeposited with the American Type Culture Collection, Washington, D.C.,on the 29th day of October 1964, and is recorded there as Number VR332.

The pig kidneys which we select for use in our process are those whichwe obtain from young pigs, including foetal pigs, delivered from the sowby Caesarian section or by natural birth, desirably from a minimaldisease herd. In this way, we are able to obtain kidney tissue which canbe maintained and grown by tissue cell techniques and which has thedesirable quality of being free from contaminants, such as hog choleraor other pig diseases.

in producing our vaccine, the ERA strain of rabies virus is passed manytimes, for example 20 times, through pig kidney cells. The titre ofvaccine produced at the fourth pig kidney passage is about 10- and atthe 20th passage the titre has increased in a most unexpected and highlysatisfying way to 10- (In speaking of titre, we refer to LD of our virusdetermined by injecting intracerebrally dilutions thereof in a quantityof 0.03 ml. per 10-15 gram mouse.) A satisfactory vaccine may beobtained from as early as the eighth passage through kidney cellswherein a titre as high as 10- was obtained. When we anticipate that wehave a high titre, we remove the fluid containing the living virus fromthe suspension of kidney cells simply by decanting the fluid therefrom.The fluid containing the virus has an extremely low content of foreignprotein thus being differentiated sharply from Semple vaccine and Fluryvaccine. Indeed, the fluid which we obtain consists essentially of thesalts and other components added in the tissue culture technique topermit the growth of kidney cells and that component which we are soanxious to achieve, namely, the ERA strain rabies virus. This fluid maybe used in undiluted form but be cause of its high titre, it may bediluted many fold with saline or other suitable diluent. We haveachieved remarkable success in the protection of cattle and otheranimals against challenge doses of rabies virus even when we havediluted our vaccine as much as IOU-fold. It may be stored for anindefinite period in frozen or dried form. The following are examples ofthe process of the invention:

EXAMPLE 1 Kidneys were removed from a pig, four to six weeks of age. Thepig was one which had been Caesarian derived. The tissue was trypsinizedfor tissue culture. Growth media used was anks and LactalbuminHydrolysate (Proceedings of the Society for Experimental Biology andMedicine, 81, 208. 1952, more specifically described as Hanks SaltSolution, Streptomycin, Phenol Red and Lactalbumin Hydrolysate 0.5%),with 5% bovine serum and 1% sodium bicarbonate plus 200 units ofpenicillin per ml. of the total volume. The media was seeded with thetrypsinized kidney cells in an amount to provide l-ml.

of packed cells per 1,000 ml. of growth media. The cells were then grownin Blake bottles, 100 m libf cell suspension being allotted to eachbottle. The bottles were incubated at 37 C. until there was a completemono layer of cells. This required a period of approximately five days.The media was then changed to a maintenance mediaHanks and lactalbuminhydrolys-ate with 2% bovine serum and 5% sodium bicarbonate plus 200units of penicillin per ml. of the total volume.

One day later, the cells were infected with rabies virus, that is, theERA strain of rabies virus otherwise known as inoculum rabies virus ATCCVR332, which had been through 15 kidney cell passages. Each bottlecontaining 100 ml. of media was inoculated with 2.5 ml. of inoculum.

On the third day, the fluid was removed, discarded and replaced by freshmedia. This was repeated on the seventh day.

Fresh media was put on the cells at the 3rd, 7th and 10th days. Theinfected fluid changes from the 3rd and 7th days were discarded. On the10th and the 14th day, the fluid changes were harvested under sterileconditions (that from the 10th day being replaced by fresh media) and aportion of each harvested fluid change was diluted with an equal volumeof a menstruum suitable for this purpose; for example, a menstruumcomprising 5% skim milk powder and 0.5% sucrose in water. The dilutedfluid changes were then freeze-dried in 14 ml. amounts.

Other aliquots from the 10th and the 14th day fluid changes were frozenwith solid carbon dioxide and later thawed. Each sample was diluted inan equal volume of sterile distilled water containing normal horse serumto a concentration of 2%, to have these samples comparable with thedried product in respect of dilution. Serial tenfold dilutions were madeusing sterile distilled water plus 2% normal horse serum as diluent. Sixmice were inoculated per dilution and each mouse received anintracerebral inoculation of 0.03 ml. The LD in mice inoculated withfluid change from the 10th and 14th day was found to be 10 and 10respectively.

After freeze-drying, the LD in mice was again determined. Two vials fromthe 10th day fluid change and two vials from the 14th day fluid changewere picked at random. Each vial was reconstituted with 14 ml. ofsterile distilled water. Using this suspension, serial tenfold dilutionswere made with sterile distilled water and 2% normal horse serum asdiluent. The LD titres in mice immediately after freeze-drying weredetermined to be l-*- for the 10th day and 10- for the 14th day.

EXAMPLE 2 Fig kidney cells were prepared as previously discribed.

In four days, the monolayer of cells was complete and the growth mediawas changed to the maintenance media.

One day later, bottles containing 100 ml. of media were inoculated with2.5 ml. of ERA strain of rabies virus, in its 29th serial pig kidneypassage, th day fluid change.

Fresh media was put on the cells at the 6th day. The fluids from the 6thand the 10th day were harvested under sterile conditions, and a portionwas diluted with an equal volume of a menstruum comprising 5% skim milkpowder and 0.5% sucrose in water. It was then freezedried in 14 ml.amounts.

Other aliquots from the 6th and the 10th day fluid changes were frozenwith solid carbon dioxide and later thawed. Each sample was then dilutedwith an equal volume of sterile distilled water plus 2% of normal horseserum, so that these samples were comparable with the dried product inrespect to dilution. Serial ten-fold dilutions were made using steriledistilled water plus 2% of normal horse serum as diluent. Six mice wereinoculated per dilution and each mouse received an intracerebralinoculation of 0.03 ml. The LD titres for mice inoculated with fluidchanges from the 6th and 10th day were calculated to be 10 and l0respectively.

Immediately after freeze-drying, the LD titres for mice were againdetermined. Two vials from the 6th day and two vials from the 10th daywere taken at random. Each vial was reconstituted with 14 ml. of steriledistilled water. From each of the 6th day vials, an equal portion wasremoved and pooled. A 10th day pool was similarly prepared. Serialten-fold dilutions of these suspensions were then prepared in distilledwater plus 2% of normal horse serum and injected into mice as previouslydescribed. The LD titres were calculated to be 10*- for the 6th day and10' for the 10th day.

The following experiments demonstrate the protection afforded by ourrabies vaccine.

Experiment 1 In this experiment a group of 15 cattle, one year old, wasdivided into three sub-groups of live cattle each. The first sub-groupreceived by intramuscular injection 3.0 ml. of our vaccine produced bytissue culture utilizing dog kidney. The second sub-group received alike dose of Experiment 2 This experiment involved 20 cattle, of oneyear of age, five groups of four cattle each, one of these groupsservingl as a control. The vaccine was produced by our method and driedfrom the frozen state. The dosage was as noted in the table below. Afterone month the animals were. challenged as in Experiment 1, using :2 ml.of challenge virus having a titre of 10* determined as before. Theresults were as follows:

Vaccine Volume N o. Survived Undiluted 3 4 4/4 1/10 dilution 3 4 4/411100 dilution 3 4 4/4 3 4 1/4 4 0/4 Thus it will be seen that ourvaccine even when diluted one-thousandfold gave some protection withpositive protection even when diluted one-hundredfold.

Experiment 3 This experiment was designed to show a use for our vaccinein the protection of sheep from rabies. In this case, the controlinvolved seven unvaccinated sheep. The vaccine was freeze-dried. Theamount injected was 3.0 ml. of the reconstituted vaccine and one monthlater all animals received intramuscularly a challenge dose of 2.0 ml.of rabies virus having a titre of 10* determined as before.

The results were as follows:

Vaccine Volume N 0. Survived Undiluted 3 5 5/5 1/10 dilution... 3 5 5/51/100 dilution 3 5 5/5 1/1,000 dilution 3 5 4/5 Controls 7 1/7 Thus itis clear that our vaccine, even when diluted onethousandfold hasafforded marked protection against 5 rabies and complete protection whendiluted one-hundredfold.

Experiment 4 V accinc Volume No. Survived Undilutcd A 2 4 4/4 1/10dilution 2 4 4/4 1/100 dilution. 2 4 4/4 1/1 ,000 dilution 2 4 3/4Controls 4 /4 Thus, it can be seen that all unvaccinated dogs diedwhereas our vaccine diluted 1:1000 gave significant pro tection andcomplete protection when diluted 1:100.

Experiment 5 Sixty-eight dogs from 2-4 months of age were vaccimatedwith our tissue culture vaccine with dosage of 2 cc. administeredintramuscularly. Two different lots of vaccine were used. Two monthslater all vaccinates and sixteen control dogs were challenged as beforewith rabies virus with the following results:

No. Survived Vacciuates 68 67/68 Controls 16 0/16 What we claim as newand desire to protect by Letters Patent of the United States is:

1. A process for the preparation of rabies virus composition adapted forintroduction into the body of an animal in order to confer immunityagainst subsequent infection by rabies virus comprising:

(a) infecting a culture of live kidney cells derived from pigs in amaintenance medium solution with ERA strain rabies virus ATCC VR332,

(b) allowing the virus to multiply in said culture for several days, and

(c) separating the kidney cells from the solution and harvesting theresulting virus-containing solution.

2. A process for the preparation of rabies virus solution according toclaim 1 in which the maintenance medium is a solution comprising Hankssalt solution and lactalbumin hydrolysate with 2% bovine serum, 5%sodium bicarbonate and 200 units of pencillin per ml. of total volume.

3. A process for the preparation of live rabies vaccine comprising:

(a) infecting a culture of viable pig kidney tissue in a maintenanceculture medium with ERA strain rabies virus ATCC VR 332,

(b) allowing the virus to multiply in said culture for several days,

(c) separating said culture medium infected With said virus from saidtissue culture,

(d) replacing said culture medium. with a fresh maintenance culturemedium,

(e) repeating steps (b), (c) and (d) until a virus infected culturemedium having an LD titre in mice of at least is obtained, and

(f) separating the tissue from the culture medium and harvesting theresulting 'virus-containing culture medium.

4. A process for the preparation of rabies virus solution adapted forintroduction into the body of an animal in order to confer immunityagainst subsequent infection by rabies virus according to claim 3comprising:

(g) repeating steps (b), (c) and ((1) until a virus infected mediumhaving an LD titre in mice of at least l0 is obtained, and

(h) separating the kidney cells from the solution obtained in step (g)and harvesting the resulting viruscontaining solution having an LD titrein mice of at least 1O 5. A process for the preparation of rabies virussolution according to claim 3 in which said high titre infectedvirus-containing solution is diluted with a menstruum comprising about5% skim milk powder and about 0.5% sucrose in Water and freeze-dryingfor storage.

6. A process for the preparation of rabies virus solution according toclaim 3 wherein steps (b), (c) and (d) are repeated at least 8 times.

7. A live rabies vaccine comprising a strain of rabies virus ATCC VVR332 in a pharmaceutically acceptable injectable fluid.

8. The vaccine of claim 7 wherein the pharmaceutically acceptableinjectionable fluid is a medium solution used in the culturer of livedog kidney cells.

9. The vaccine of claim 7 wherein the pharmaceutically acceptableinjectionable fluid is a medium solution used in the culture of live pigkidney cells.

10. A process for the preparation of live rabies vaccine comprising thesteps of introducing ERA strain rabies -virus ATCC VR 332 into a tissueculture maintenance medium containing viable cells of dog kidney tissue,incubating said tissue culture medium by tissue culture techniques,removing the kidney cells and then harvesting the resulting rabiesvirus-containing solution.

11. A process for the preparation of live rabies vaccine comprising thesteps of introducing ERA strain rabies virus ATCC VR 332 into a tissueculture maintenance medium containing viable cells of pig kidney tissue,in-

cubating said tissue culture medium by tissue culture techniques,removing the kidney cells and then harvesting the resulting rabiesvirus-containing solution.

12. A process for the preparation of live rabies vaccine comprising:

(a) infecting a culture of viable dog kidney tissue in a maintenanceculture medium with ERA strain rabies virus ATCC VR 332;

(b) allowing the virus to multiply in said culture for several days;

(c) separating said culture medium infected with said virus from saidtissue culture;

(d) replacing said culture medium with a fresh maintenance culturemedium;

(e) repeating steps (b), (c) and (d) until a virus infected culturemedium having an LD titre in mice of at least 10 is obtained; and

(f) separating the tissue from the culture medium and harvesting theresulting virus-containing culture medium.

References Cited Abelseth: (I), Canadian Veterinary Journal, vol. 5, No.4, pp. 84-87, April, 1964, 167-78 vk.

Abelseth: (II), Canadian Veterinary Journal, vol. 5. No. 11, pp.279-286, Nov. 1, 1964, 167-78 vk.

RICHARD L. HUFF, Primary Examiner.

US. 01. X.R. -11, 1.2, 1.3

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3 ,423,505 Dated Q [many 2] 959 & Inventor)? John F. Crawley and Me1vin K.Abelseth It is certified that error appears in the above-identifiedpatent and that said Letters Patent are hereby corrected as shown below:

De1ete the fi rst "V" in 1 ine 2 of c1aim 7.

Signed and sealed this 11th day of February 1975.

(SEAL) Attest:

C. MARSHALL DANN RUTH C. MASON Commissioner of Patents Arresting Officerand Trademarks

